The present invention relates to novel isolated proteases of the RP-II type and variants of RP-II proteases exhibiting improved properties in comparison to the parent RP-II protease, DNA constructs and vectors coding for the expression of said proteases and variants, host cells capable of expressing the proteases and variants from the DNA constructs, as well as a method of producing them by cultivating said host cells. The proteases may advantageously be used as constituents in detergent compositions and additives.
Proteases of the subtilisin family (Siezen et al. Protein Science 6 (1997) 501-523) have for many years been used in the detergent industry due to their apparent superiority over other protease types in this type of application.
A large number of subtilisins and the related subtilases are known.
Protease variants have been produced in a number of subtilisin proteases in order to provide changes in various properties, such as thermo stability, specific activity, pH-dependency, isoelectric point, wash performance, oxidation stability, autoproteolysis, etc.
Such variants are disclosed in various patent publications, such as EP 130 756, EP 251 446, and EP 824 585.
The fact that novel detergents constantly are being developed to satisfy various user demands provides an incentive to continuously develop novel proteases capable of providing excellent performance in the novel detergents.
Bacillus proteases of the RP-II type are another type of serine proteases that in primary structure are similar to chymotrypsinogen.
The first description of a protease of the RP-II family of Bacillus proteases was in U.S. Pat. No. 4,266,031 (Tang et al., Novo Industri A/S), where it was designated Component C and tentatively characterised as not being a serine protease or metalloprotease. Component C was considered a contaminant in the production of the Bacillus licheniformis alkaline protease, subtilisin Carlsberg.
In EP 369 817 (Omnigene Bioproducts, Inc.) the B. subtilis member of the RP-II family was identified by its amino acid and DNA sequences. The enzyme was stated not to be a serine protease, and the family name RP-II designated (Residual Protease II). The enzyme was characterised further by the inventors of EP 369 817 in Rufo et al., 1990, J. Bacteriol. 2 1019-1023, and Sloma et al., 1990, J. Bacteriol. 2 1024-1029, as a metalloprotease designating the enzyme as mpr.
WO 91/13553 (Novo Nordisk A/S) disclosed the amino acid sequence of the C component now stating that it is a serine protease specific for glutamic and aspartic acid , while EP 482 879 (Shionogi and Co. Ltd.) disclosed the enzyme and a DNA sequence encoding the C component from B. licheniformis ATCC No. 14580, naming the enzyme BLase. In EP 482 879 the protease is described as being specific for glutamic acid.
In 1997 Okamoto et al. (Appl. Microbiol. Biotechnol. (1997) 48 27-33) found that the B. subtilis homologue of BLase, named BSase was identical to the above mentioned enzyme, mpr/RP-II.
Initial testing of the B. licheniformis member of the RP-II family had indicated that this enzyme in some aspects may be inferior in detergents in comparison to the subtilisins.
However, it is believed that a screening program for novel RP-II family members both isolated from nature (wild types) and recombinantly produced variants thereof will provide alternative proteases for use in detergents.
Consequently it is an object of the present invention to provide novel RP-II protease members obtainable from various Bacillus strains.
Furthermore it is the object of the present invention to design novel variants of the RP-II proteases having improved properties as compared to those of their parent protease.
Accordingly, in a first aspect the present invention relates to novel isolated RP-II proteases selected from the group consisting of:
(i) a RP-II protease that is immunochemically identical or partially identical by cross-reaction with an antibody raised against or reactive with at least one epitope of a RP-II protease comprising the amino acid sequences of the mature peptides shown in the appended Sequence Listing SEQ ID NO: 2, 4, ID No. 6, ID No. 8, ID No. 10, or ID No. 12; and/or
(ii) a RP-II protease that is at least 60% homologous with the amino acid sequence of a RP-II protease comprising the amino acid sequence shown in the appended Sequence Listing SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, or SEQ ID NO: 12; and/or
(iiia) a RP-II protease that is encoded by a DNA sequence which hybridizes with an oligonucleotide probe hybridizing with a DNA sequence encoding a RP-II protease comprising the amino acid sequence shown in the appended Sequence Listing SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, or SEQ ID NO: 12; and/or
(iiib) a RP-II protease that is encoded by a DNA sequence which hybridizes with an oligonucleotide probe hybridizing with a DNA sequence encoding a RP-II protease comprising the DNA sequence shown in the appended Sequence Listing SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, or SEQ ID NO: 11;
(iv) an allelic variant of (i), (ii), or (iiia or iiib);
(v) a subsequence of (i), (ii), (iiia or iiib), or (iv), wherein the subsequence has protease activity.
The invention furthermore relates to RP-II protease variants produced by modifying at least one amino acid residue within the mature enzyme in order to obtain a modification of the properties of the parent enzyme.
Such variants of the present invention are contemplated to have improved substrate specificities, catalytic rate, stability, especially towards the action of proteolytic enzymes and/or detergent ingredients, thermostability, storage stability, improved resistance towards peroxidase/pHBS inactivation, and/or improved wash performance as compared to the parent RP-II protease. The variants of the invention include fragments of the RP-II proteases or variants thereof having retained protease activity.
The present invention also relates to novel isolated nucleic acid sequences encoding RP-II proteases, selected from the group consisting of:
(a) a nucleic acid sequence having at least 60% homology with the nucleic acid sequence encoding the mature polypeptide of SEQ ID NO: 1, 3, 5, 7, 9, or 11;
(b) a nucleic acid sequence which hybridizes under low stringency conditions with (i) the nucleic acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, or 11 (ii) the cDNA sequence of SEQ ID NO: 1, 3, 5, 7, 9, or 11, (iii) a subsequence of (i) or (ii) of at least 100 nucleotides, or (iv) a complementary strand of (i), (ii), or (iii);
(c) an allelic variant of (a), or (b);
(d) a subsequence of (a), (b), or (c), wherein the subsequence encodes a polypeptide fragment which has protease activity; and
The present invention also relates to nucleic acid or DNA constructs comprising a DNA sequence encoding a RP-II protease or RP-II protease variant as indicated above, recombinant expression vectors carrying said DNA construct, cells transformed with a DNA construct or expression vector, as well as methods for producing a RP-II protease or variant of the invention by culturing or growing said cell under conditions conducive to the production of the protease or variant, after which the protease or variant is recovered from the culture, and optionally purified to be substantially pure.
The invention further relates to an enzyme granulate, a liquid enzyme composition or a protected enzyme preparation comprising a RP-II protease or protease variant of the invention and suitable for the preparation of e.g. a detergent composition comprising a RP-II protease or RP-II protease variant of the invention.
Deposited Biological Materials
DNA encoding the novel RP-II proteases of the invention has been inserted into plasmids used to transform E. coli. These transformants have been been deposited according to the Budapest Treaty on the International Recognition of the Deposits of Microorganisms for the Purpose of Patent Procedures, on Dec. 3, 1990 at DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM), Mascheroder Weg 1b, D-38124 Braunschweig, Germany, under Accession Nos. DSM 12841 (AC116), DSM 12842 (CDJ31), DSM 12843 (BO32), DSM 12844 (JA96), and DSM 12845 (AA519).
The deposits have been made under conditions that assure that access to the culture will be available during the pendency of this patent application to one determined by the Commissioner of Patents and Trademarks to be entitled thereto under 37 C.F.R. xc2xa71.14 and 35 U.S.C. xc2xa7122. The deposit represents a substantially pure culture of the deposited strain. The deposit is available as required by foreign patent laws in countries wherein counterparts of the subject application, or its progeny are filed. However, it should be understood that the availability of a deposit does not constitute a license to practice the subject invention in derogation of patent rights granted by governmental action.